Single cell analysis in head and neck cancer reveals potential immune evasion mechanisms during early metastasis

Profiling tumors at single-cell resolution provides an opportunity to understand complexities underpinning lymph-node metastases in head and neck squamous-cell carcinoma. Single-cell RNAseq (scRNAseq) analysis of cancer-cell trajectories identifies a subpopulation of pre-metastatic cells, driven by actionable pathways including AXL and AURK. Blocking these two proteins blunts tumor invasion in patient-derived cultures. Furthermore, scRNAseq analyses of tumor-infiltrating CD8 + T-lymphocytes show two distinct trajectories to T-cell dysfunction, corroborated by their clonal architecture based on single-cell T-cell receptor sequencing. By determining key modulators of these trajectories, followed by validation using external datasets and functional experiments, we uncover a role for SOX4 in mediating T-cell exhaustion. Finally, interactome analyses between pre-metastatic tumor cells and CD8 + T-lymphocytes uncover a putative role for the Midkine pathway in immune-modulation and this is confirmed by scRNAseq of tumors from humanized mice. Aside from specific findings, this study demonstrates the importance of tumor heterogeneity analyses in identifying key vulnerabilities during early metastasis.

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Policy information about availability of of computer code Data collection Data analysis N Gopalakrishna Iyer Mar 13, 2023 All code used to to analyse the dataset is openly available at at https://doi.org/10.5281/zenodo.7692887. All software and algorithms used in in this study are publicly available and are listed in in the Methods section.
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Further details can be found in the Methods of the manuscript.
The raw human and mouse 10x scRNAseq raw data generated in this study and the corresponding processed Seurat objects with all cells have been deposited in the GEO under accession code GSE188737 We did not perform or report analysis based on sex and gender in our study as this information was not relevant to the study. Both females and males were used in our dataset without any prejudice.
For tumor samples, covariant-relevant population characteristics including clinical and pathologic features are provided in Supplementary data 1 and 2; subjects included both males and females, aged 21-85.
Umbilical cord blood samples were collected from uncomplicated pregnancies at term and patients were recruited from planned Cesarean deliveries without any prejudice.
For tumor samples, all patients were confirmed histologically to be HNSCC and suitable for surgical resection (with no prior cancer treatment).
Umbilical cord blood samples were collected from uncomplicated pregnancies at term and patients were recruited from planned Cesarean deliveries without any prejudice.

March 2021
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Single cell and bulk sequencing data from patients were not suitable for replication. For functional experiments, at least n=2 of data point was performed from at least 2 independent experiments and all attempts at replication were successful.
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All cell lines were patient-derived tumor cell cultures that were passaged until a majority of tumor cells was observed.
Cell line identity was authenticated by comparing the STR profile (Indexx BioResearch), mutational and/or expression profile of each cell line to its original tumour.
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All cell lines were derived directly from patient tumors and passaged until a majority of tumor cells was observed. No genetic manipulation was performed.
Six to eight weeks old NOG-EXL (hGM-CSF/hIL-3 NOG) female mice (n=16) were used for human CD34+ hematopoietic stem cells preengraftment. Sixteen weeks later, these mice were treated with or without a course of Pembrolizumab. Mice were housed in a 12 light/12 dark cycle at approximately 18-23 degree Celsius with 40-60% humidity.
The study did not involve wild animals.
We did not perform or report analysis based on sex and gender as this information was not relevant to the study. Only female mice were available and used for the experiment.
The study did not involve samples collected from the field.
All animal experiments were approved by the Institutional Animal Care and Use Committee of the Biological resource centre (BRC), A*STAR, Singapore (IACUC numbers 161192, 191496) nature portfolio | reporting summary

March 2021
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Methodology
Sample preparation Instrument Software Cell population abundance Gating strategy Tick this box to to confirm that a figure exemplifying the gating strategy is is provided in in the Supplementary Information.
For cell surface staining, patient-derived cell lines were trypsinized into single cell suspensions and cultured PBMCs were stained with Fixable Viability Dye eFluor 506 (cat. #65-0866-14, eBioscience) and fluorochrome conjugated antibodies for 30-60 mins on on ice in the dark. Washes were performed using ice-cold 1% 1% BSA in in PBS.
For intracellular staining, trypsinized cells were first stained with Fixable Viability Dye eFluor 506 (cat. #65-0866-14, eBioscience) and antibodies recognizing surface antigens, and subsequently fixed and permeabilized with an an eBioscience Foxp3/Transcription Factor Staining Buffer Set according to to the manufacturer protocol. All staining steps were performed for 30-60 mins on on ice in the dark.
All samples were acquired using a BD BD FACSCanto II II or or BD BD LSRFortressa, and sorted using a BD BD FACSAria III.
Flow cytometry data was analyzed using Flowjo.
Post-sort purities were assessed by by flow cytometry and confirmed to to be be > 95%. Post-sort purities were also validated by by invasion assays For the cell line experiment, cells were gated on on FSC-A/SSC-A, and then gated to to exclude doublets based on on FSC-A/FSC-H. Dead cells were excluded based on on Fixable Viability Dye eFluor 506 (cat. #65-0866-14, eBioscience). Cellular expression of of AXL or or AURKB were categorized as as high, middle or or low/negative. An An example of of the gating strategy used is is shown in in Supplementary Figure 3J.